In previous work in collaboration with Dr. Minoru Ko (LG-NIA),we were able to establish a stem-cell differentiation assay based on cell morphology alone, using phase-contrast imaging without specific markers. An important validation of this approach was that we were able to establish conditions where cell lines showed consistent grouping by gene function over several independent experiments. Additionally, we were able to expand this analysis to compare the morphologies of 56 cell lines. These results are being prepared for publication. In preliminary work, we have been using this assay to characterize early events in the osteogenesis/adipogenesis pathway. The balance of osteogenic/adipogenic cells changes during aging, and this differentiation pathway may play an important role in age-related bone loss as well as accumulation of fat in bone marrow. We have continued work characterizing the molecular basis of morphological age-state transitions during the C. elegans life-span. In published work we used WND-CHARM to identify distinct morphological aging states in C. elegans. We used this technique to sort worms based on their age state during a transition period where an aging population is evenly divided between individuals in Stage I and Stage II. The worms were identical genetically, by chronological age, by growth conditions, and by visual appearance, and could only be sorted into age-states using WND-CHARM. Micro-array experiments performed on these two sub-populations revealed several hundred genes with significantly altered expression profiles. By comparing our gene lists with those from other aging studies in C. elegans, we were able to identify several gene families and functional groups that were unique to our study. A prevailing theme of the aging genes uniquely identified in this study were those involved in targeted proteolysis, which appears to be a hallmark of this first aging state transition. This study appeared in the journal AGE. Ongoing studies of these genes with RNAi have confirmed that some of them have aging effects. Additionally, in the coming year, the microarray experiments will be expanded to study the StateII/StateIII transition as well as characterising the genes that are differentially expressed early vs. late in these aging states.